1. Field of the Invention
The present invention relates to a new method of isolating salicylate hydroxylase from Pseudomonas bacteria and to methods of using this enzyme for the identification and quantitation of salicylate in body fluids.
2. Description of the Prior Art
The unique property of controlling pain, fever, and inflammation has made acetylsalicylic acid (aspirin) one of the most widely used drugs today. However, its frequent use and easy availability also have made it the cause of more cases of accidental poisoning in children than any other substance.
When aspirin is ingested, it ionizes and rapidly loses its acetyl group to become salicylate. Therefore, what is monitored in aspirin therapy or intoxication is salicylate; aspirin itself is analyzed seldomly because its level in patients has little clinical significance. In order for aspirin treatment to be effective, serum or plasma levels of salicylate must be kept within the therapeutic range (5-40 mg/dl serum/plasma). If aspirin is present in an overdose (over 45 mg/dl serum/plasma level), the resulting high level of salicylate acts as a serious poison often causing coma and death.
Since salicylate intoxication occurs frequently, it would be very useful to develop a simple, quick, and specific method for identification as well as quantitation of salicylate in patients who are taken to an emergency room for an overdose of an unknown drug. Such a method would also be valuable in routine monitoring of salicylate levels in those patients who require continuous aspirin therapy. Unfortunately, no test having all the desirable characteristics now exists.
Presently, the most popular analytical methods for salicylate are gas chromatography (GC), liquid chromatography (LC), and the Trinder method. Because of several obligatory steps, such as deproteinization, extraction, derivatization, and column regeneration, that must be carried out for each analysis, GC and LC are laborious and time consuming. As such, they are not the methods of choice in an emergency situation when quick identification of the poison followed by proper treatment is essential. In addition, they require expensive instruments (which are also costly to maintain) and specially trained technicians.
The Trinder method, which is based on the development of a purple color as a result of the reaction between Fe.sup.+++ and compounds having an enolizable hydroxyl group, such as phenols (salicylate is a phenol), is simple and relatively quick but, unfortunately, is not salicylate-specific. Thus the Trinder test is severely interfered with not only by hundreds of phenols but also by compounds like acetoacetate (found in diabetic patients). In addition, phenothiazines, which have intoxicating incidents slightly more frequent than salicylate, give a color that can be easily mistaken for that caused by salicylate. In fact, the same ferric reagent can be employed for the detection of both salicylate and phenothiazines. The spectra of several products of the Trinder reaction with compounds that interfere with the quantitation of salicylate are shown in FIGS. 1A) and B). Because of these frequent false-positive reactions, the Trinder method is always accompanied by the danger of misdiagnosis.
Furthermore, the Trinder method often give irrational results if the serum/plasma salicylate concentration is below 10 mg/dl. The serum from a patient who never received salicylate often shows a salicylate level as high as 10 mg/dl. Such irrational results are most likely due to turbidity caused by serum proteins under acidic condition (the Trinder reagent is made in HCl, HNO.sub.3, or H.sub.2 SO.sub.4). The manufacturers of the Trinder reagent claim that the turbidity can be removed by centrifugation at 3,000 rpm for 10 minutes. In the experience of the present inventor, however, slight turbidity remains after centrifugation at this speed when a centrifuge of the type commonly available in toxicology and clinical laboratories is used (Table-top type). Complete removal of the turbidity can be achieved if centrifugation is carried out at a 100,000xg for 15 minutes, but centrifuges capable of the requisite high speeds are less common in the clinical laboratory. Some medical centers formerly employed the Trinder method but, because of persistent inconsistent results, now extract salicylate with ethylene dichloride from serum before subjecting the serum to the Trinder test; addition of the extraction step makes the Trinder test no longer a fast method and its greatest advantage is lost.
Another desirable feature for a new salicylate test method would be compatability with an automatic analyzer. Many toxicology and clinical chemistry laboratories which handle large numbers of samples are equipped with automatic analyzers. No method currently available can use these analyzers for accurate salicylate determinations because of the turbidity problem. Adoption of the Trinder method to auto analyzers is possible if the most time consuming step, namely removal of the turbidity by centrifugation or by filtration, is performed manually. However, if this step is carried out manually, it is senseless to use an automatic analyzer since manual determination of the absorbance of the clarified samples by a spectrophotometer is simpler and less expensive. It should also be pointed out that the use of the Trinder reagent in an expensive automatic analyzer is risky since the reagent is corrosive and contains high concentrations of heavy metals such as Fe and Hg or W; contamination of the expensive instrument by these metals would cripple the analyzer's functions that involve enzymes because even trace amounts of these metals often serve as potent inhibitors of many enzymes.
In 1970, White-Stevens and Kamin reported the purification and properties of salicylate hydroxylase (salicylate 1-monooxygenase EC 1.14.13.1) from a soil microorganism (Biochem. Biophys. Res. Commun. 38, 882-889 (1970)) later identified as a Pseudomonas. This enzyme catalyzes the unidirectional conversion of salicylate to catechol in the presence of molecular oxygen and NAD(P)H (nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate). Kamin's enzyme is physically as well as catalytically different from the salicylate hydroxylase that was isolated from Pseudomonas pupita and described by Yamamoto et al in Japan five years earlier (J. Biol. Chem., 240, 3408 (1965)). Thus, Kamin's enzyme has a molecular weight of 91,000.+-.3000 and is composed of two apparently identical subunits, each of which contains one FAD (flavin adenine dinucleotide) and can utilize both NADH and NADPH (with the same V.sub.max) as reductant. On the other hand, the enzyme purified by the Japanese workers is a monomeric protein containing one FAD per molecular mass of 57,200 daltons and does not utilize NADPH. Nevertheless, similarities exist between these enzymes as would be expected for enzymes isolated from bacteria within the same genus that perform the same physiological function. Because these salicylate hydroxylase enzymes require an external reductant (i.e., NADPH or NADH) and only one oxygen atom of the molecular oxygen is incorporated into the substrate as a hydroxyl group during the catalysis, they belong to the class of enzymes known as external flavoprotein monooxygenases.
It would be desirable to have an enzymatic method of determining salicylate levels in body fluids since such a method would be specific for salicylate in the presence of more interfering substances than are now allowed with current test methods and would allow more rapid determination of salicylate level than LC or GC methods. In order to make such a method readily available, it is also desirable to have a method of purifying the chosen enzyme in an adequate yield and to the desired level of activity. However, prior to the present invention, no such methods existed.